1. Field of the Invention
This invention relates to a method for determining the presence or amount of analyte in a test sample using magnetically responsive materials. More particularly, the invention relates to the use of magnetically responsive materials to change the properties of components in binding assays.
2. Discussion of the Art
Diagnostic assays have become an indispensable means for detecting analytes in test samples by using the mutual reaction between the analyte and a specific binding member for the analyte, such as the immunoreaction between an antigen and an antibody that binds to that antigen. Typically, detectable tags or labels attached to antibodies, which in turn bind to the analyte of interest, are employed in such diagnostic assays, wherein the detection of the resultant labeled antibody-analyte complex, or detection of the labeled antibody that does not bind to the analyte to form a complex, is used to indicate the presence or amount of analyte in the test sample.
Two commonly used diagnostic assay techniques employing specific binding members are the radioimmunoassay (RIA) and the enzyme immunoassay (EIA), both of which employ a labeled specific binding member. The RIA uses a radioactive isotope as the detectable tag or label attached to a specific binding member. Because the radioactive isotope can be detected in very small amounts, it can be used to detect or quantify small amounts of analyte. However, substantial disadvantages associated with the RIA include the special facilities and extreme caution that are required in handling radioactive materials, the high costs of such reagents, and their unique disposal requirements.
The EIA uses an enzyme as the detectable tag or label attached to a specific binding member, wherein the enzymatic activity of the enzyme is used to detect the immunoreaction. While the EIA does not have some of the same disadvantages of the RIA, EIA techniques typically require the addition of substrate materials to elicit the detectable enzyme reaction. In addition, enzyme substrates are often unstable and have to be prepared just prior to use or be stored under refrigeration. Moreover, enzyme labels may be difficult to purify and conjugate to binding members, and may be unstable during storage at room temperature or even under refrigerated conditions. Enzyme immunoassays are also unsatisfactory in that the methods typically require complex incubations, multiple liquid additions, and multiple wash steps.
More recently, assay techniques using metallic sol particles as visual labels have been developed. In these techniques, a metal (e.g., gold, silver, platinum), a metal compound, or a nonmetallic substance coated with a metal or a metal compound, is used to form an aqueous dispersion of particles. Generally, the specific binding member to be labeled is adsorbed onto the metallic sol particles, and the particles are captured or aggregated in the presence of analyte. Although the metallic sol particles have the advantage of producing a signal that is visually detectable as well as measurable by an instrument, they are difficult to measure quantitatively. The metallic sol particles also have a limited color intensity, and consequently, limited sensitivity in some assays. In addition, the surfaces of inorganic metallic sol particles, such as gold, may not readily accept the covalent attachment of specific binding members. Thus, during use in a binding assay, care must be taken so that the adsorbed specific binding members are not removed from the inorganic particles through the combination of displacement by other proteins or surface active agents and the shear forces that accompany washing steps used to remove non-specifically bound material. Metallic sol particles can be difficult to coat without inducing aggregation; they may aggregate upon storage or they may aggregate upon the addition of buffers or salts. Furthermore, such particulate labels are difficult to concentrate and can be difficult to disperse.
Other materials for labels include chemiluminescent and fluorescent substances. However, these substances can be unstable, and fluorescent materials may undergo quenching. Non-metallic particles, such as dyed or colored latex particles and selenium particles, have also been used as visual labels.
Self-performing immunoassay devices have proven to be of great benefit in the field of diagnostics. A self-performing immunoassay device is a kit containing immunoreagents to which a biological sample can first be added by the patient or laboratory technician, then the diagnostic assay performed without the need for complex laboratory instruments. Commercially available self-performing immunoassay devices, such as the strip assay device having the trademark "TESTPACK PLUS", distributed by Abbott Laboratories, enable immunoassays to be performed quickly and reliably.
Typically, self-performing immunoassay devices involve chromatographic test strips. For example, U.S. Pat. No. 4,960,691 discloses a test strip for analysis of an analyte in a sample by means of a sequential series of reactions. The test strip comprises a length of chromatographic material having capillarity and the capacity for chromatographic solvent transport of non-immobilized reagents and reactive components of a sample by means of a selected chromatographic solvent. The test strip includes (1) a first end at which chromatographic solvent transport begins, (2) a second end at which chromatographic solvent transport ends, and (3) a plurality of zones positioned between the first and second ends. These zones include (1) a first zone impregnated with a first reagent which is mobile in the solvent and capable of a specific binding reaction with the analyte, (2) a second zone for receiving the sample, and (3) a third zone, downstream of the second zone, impregnated with a second reagent that is immobilized against solvent transport and is capable of a specific binding reaction with the analyte so as to immobilize the analyte in the third zone. The test strip is designed so that the first reagent can be detected at the third zone as a measure of the analyte.
A common feature of chromatographic test strips involves the flow of a fluid or a mixture of a fluid and particles through a porous matrix. The test strip typically includes a reaction zone where binding reactions can occur. For proper binding reactions to occur in chromatographic test strips, the fluid or mixture must flow substantially uniformly through the reaction zone.
A problem with assay devices of this type is the inherent variability in the material from which the porous matrix is formed. This variability (for example, in porosity) directly affects the flow of fluid through the matrix and may adversely affect the precision of the assay device. Furthermore, the matrix will often non-specifically bind the particles or reagents at sites at the intended reaction zone or elsewhere, thereby necessitating the use of elaborate passivating procedures after the immobilized reagent has been applied. Consequently, there is a desire to develop a rapid, simple, self-performing assay device that does not require a fluid to flow through a porous matrix.
Another problem with self-performing immunoassay devices is the necessity of immobilizing a specific binding reagent on the test strip so that reagents involved in the assay can be captured at the reaction zone. The process of immobilizing the specific binding reagents on the test strip can be difficult to control, leading to lot-to-lot variations in the binding capacity of the reaction zone. Furthermore, the immobilized binding reagents can be unstable, causing the binding capacity of the reaction zone to change after shipping or storage. Because the immobilized specific binding reagent is specific for the assay of a particular analyte, test strips must be dedicated to a particular assay. An additional problem with self-performing immunoassay devices is lot-to-lot variation resulting from manufacturing processes, especially variation of the activity of the biological reagents, such as the binding molecules. For example, lot-to-lot variations in the binding capacity of the binding reagent at the capture zone of a test strip can affect assay results. Although adjustments in the activities or concentrations of the other reagents can compensate, making such adjustments involves introducing undue complexity to the manufacturing process and necessitates matching each lot of test strips to particular lots of reagents. The ability to use a completely stable, uniform test strip in assays for several different analytes would greatly simplify the production and control of strip-based self-performing assays. Alternatively, the ability to readily adapt a test strip during manufacturing to meet the requirements of a set of reagents would be advantageous.
In several applications it is desirable to use a self-performing assay which gives a positive result above a certain analyte concentration and a negative result below that concentration, with a very narrow range of transition concentrations. This result has been difficult to achieve with conventional test strips.
Superparamagnetic microparticles are also used extensively in the performance of immunoassays. Superparamagnetic microparticies are magnetically responsive in that an applied magnetic field will cause a force to act upon them in the direction of the magnetic field generator. However, they will not retain any residual magnetism after the applied magnetic field is removed. Typically, the particles are attached to a specific binding member to form a conjugate, the specific binding member being capable of binding to an analyte of interest. The specific binding member-particle conjugate is dispersed in a liquid, which is then mixed with the sample to form a test mixture, thereby allowing the specific binding member-particle conjugate to bind the analyte, if analyte is present. The conjugate-analyte complex is then attracted to a solid surface by the application of a magnetic field and the material not bound to the conjugate is removed (commonly known as bound/free separation), as described in U.S. Pat. Nos. 4,745,077; 4,070,246; and 3,985,649. Additional wash steps, reagent additions, and bound/free separations are usually required before a measurable signal is produced. Analytical methods of this type typically use light emission (chemiluminescence or fluorescence), light absorption after the enzymatic production of a chromophore, or radioactive emission as the signal indicative of the amount of the analyte of interest. Typically, the magnetic responsiveness of the superparamagnetic particles is used only as an aid in the bound/free separation steps, with the remainder of the assay procedure involving conventional reagents and protocols. Consequently, conventional analyses using superparamagnetic particles are limited to either complex automated instrumentation (for example, the ACS 180 from Ciba Corning Diagnostics) or an extended series of manual assay steps.
The size and composition of the superparamagnetic particles and the strength and gradient of the applied magnetic field will determine the magnitude of the magnetic force exerted upon them. When a magnetic field is applied to a liquid suspension of such particles, the magnitude of the force exerted on each particle, and the hydrodynamic drag of each particle, will determine its rate of movement through the liquid toward the magnetic field generator. For magnetically responsive particles of similar composition, the force exerted upon an individual particle by an applied magnetic field, and hence its rate of movement through the liquid, depends upon its volume, while drag is determined by its cross-sectional area. Smaller magnetically responsive particles will move more slowly in an applied magnetic field because of the weaker force exerted upon each particle relative to its cross-sectional area, and very small superparamagnetic particles such as ferrofluids will move very slowly because the force exerted on them is comparable to that of the random forces of the molecules surrounding them. These random forces result from thermal (Brownian) motion. As particles increase in size, their volume increases more rapidly than does their cross-sectional area, with the result that magnetic force increases more rapidly than does drag. The assembly of several small, slowly moving particles into aggregates will result in the sum of the forces acting upon the individual particles being exerted upon the aggregates, with the result that the aggregates will move more quickly through the liquid toward the source of the magnetic field than will the individual particles. The strength and gradient of the applied magnetic field can also be selected to favor the movement or capture of particular types or forms of magnetically responsive reagents.
U.S. Pat. No. 5,108,933 discloses a method whereby colloidal, magnetically responsive particles can be used for the separation of any one of a variety of target substances from a test medium suspected of containing the substance of interest through conversion of particles to micro-agglomerates including the target substance, via manipulation of their of their colloidal properties. The resultant agglomerates can subsequently be removed from the medium using ordinary laboratory magnets, as the particles are comprised of sufficient magnetic material, above an empirical threshold, to effect such removal. The method is carried out by adding to the test medium agglomerable and resuspendable colloidal particles, which are capable of stable suspension in the test medium, forming a magnetic agglomerate comprising the colloidal particles and any target substance present in the test medium, and separating the resulting magnetic agglomerates from the medium. This method of analysis, however, uses only a single type of particle, thereby presenting difficulties in detection. The presence or absence of aggregated magnetic particles in the vicinity of the magnet is neither easily nor precisely determined by visual means. It would be desirable to use indicator particles which could easily and accurately be detected visually.
The use of non-magnetic indicator particles is described by U.S. Pat. No. 5,374,531, which discloses the simultaneous use of magnetic particles and non-magnetic, fluorescent particles in the quantification of leukocyte phenotypes or other particulate analytes. Both the magnetic particles and the non-magnetic, fluorescent particles contain binding substances that bring about formation of rosettes consisting of magnetic particles, non-magnetic fluorescent particles, and the desired cells. The rosettes are separated from the non-magnetic components of the test sample by application of a magnetic field, whereupon the number of cells can be measured by the amount of fluorescence emitted by the non-magnetic, fluorescent particles. Rosette formation is applicable only to the detection of particulate analytes (such as cells), as it entails binding magnetic particles and indicator particles around the target cells, which cells must be of similar or greater size than the magnetic particles and the indicator particles. The rosettes described in this patent cannot be formed with molecular-scale analytes, as such analytes are much smaller than the magnetic particles and the indicator particles
The aggregation of magnetic and non-magnetic indicator particles as a function of the presence of molecular-scale analytes is described in U.S. Pat. No. 5,145,784. In this patent, magnetic particles and nonmagnetic detectable particles which have antigen and/or antibody affixed to their surfaces are combined with the sample to be analyzed, free antibody if required, and any necessary buffers, salts, and other reagents. After incubation for a specific time and under conditions appropriate for antigen and specific antibody to bind, the magnetic particles are removed by attraction to a magnet. The presence or absence and/or quantity of nonmagnetic detectable particles is subsequently determined and the presence or absence and/or quantity of antigen or antibody of interest in the sample is determined. In this process, the presence of analyte is not detected by directly observing the separated magnetic/nonmagnetic particle complexes near the location of the magnet.
U.S. Pat. Nos. 5,445,970 and 5,445,971 describe the use of a magnetically-attractable material as a detectable label in binding assays. The magnetic label is subjected to a magnetic field and the label, in turn, displays a resultant force or movement as a result of the application of the magnetic field. The extent of the force or movement is modulated by an analyte that may be present in a test sample. Because the presence or amount of analyte in a test sample is responsible for the magnitude of the force exerted or the amount of movement displayed by the magnetically-attractable material, the effect of the magnetic field on the magnetically-attractable label can be used as a measure of the presence or amount of analyte in a test sample. This approach requires that the presence of an analyte cause a change in the degree of binding of the magnetically-attractable material to a solid phase such that the bound magnetically-attractable material is prevented from moving in an applied magnetic field. Application of a magnetic field then causes a partitioning of the free magnetically-attractable material and the magnetically-attractable material bound to the solid phase. Measurement of the force exerted on the magnetically-attractable material bound to the solid phase, or on the free magnetically-attractable material, then reflects the quantity of analyte present in the test mixture. Although self-performing assay formats are possible using this approach, specific capture on some form of non-mobile solid phase is required.
For some applications, an assay format using only mobile solid phases such as microparticles would have distinct advantages, as would formats that do not require the measurement of magnetic force to determine analyte concentration. It would also be advantageous to utilize reagents that will not settle out of suspension. Latex particles that form stable suspensions can be produced, but superparamagnetic particles small enough to form stable suspensions, called ferrofluids, are only weakly attracted to the source of a magnetic field and therefore cannot be readily captured magnetically. Ferrofluids also are usually not compatible with aqueous solutions. It would be advantageous to develop self-performing immunoassay formats that do not require a chromatographic material. It would also be advantageous to develop a medium for a self-performing immunoassay that could be used for a multiplicity of immunoassays and easily adapted to reagent variations resulting from manufacturing processes.